This protocol allows fabrication of bone analogs without requiring heating or toxic chemicals. In this way, bone structures are formed with the potential for repairing a bone defect in clinical settings using a patient's own cells. The main advantage of the technique is that the bone ink can be printed with living cells to form any complex bone-like structure and to direct differentiation toward bone forming lineage.
Further, drugs can be loaded into the bone-ink to simultaneously enhance wound healing, bone regeneration, and to treat pathologies like cancer. Demonstrating the procedure will be Gagan Jalandhra, a PhD candidate in my lab, and Dr.Sara Romanazzo, who has worked with me and Dr.Jalandhra on various projects, including development of this technology. Begin by adding the calcium hydrogen phosphate and calcium carbonate powder mixture to a zirconia crucible such that it is no more than 75%full.
Transfer the crucible to a furnace, heat it to 1, 400 degrees Celsius at a rate of five degrees Celsius per minute and hold for three hours. Quench the reaction by removing the crucible from the furnace and leaving at a top of refractory block. Allow it to cool completely before handling.
Use a mortar and pestle to break and grind the alpha-tricalcium phosphate cake such that the resulting granules have a maximum size of 200 micrometers. Add three millimeter yttria-stabilized zirconia balls to the grinded mixture, followed by 100%ethanol. Secure the lid and grind for two hours at 180 rotations per minute.
Collect the suspension and separate the balls, using 100%ethanol for washing. Dry the suspension in an oven at 120 degrees Celsius for 24 hours. Add the dried powder to the milling jars with one millimeters zirconia balls and 100%ethanol in the same weight ratios as in the first stage.
Grind for two hours at 180 rotations per minute. Then separate and dry in the oven. Remove the sample from the oven.
To make the bone-ink, add 630 microliters of glycerol and 130 microliters of polysorbate 80 to a ball mil jar. Then add 100 milligrams of ammonium phosphate dibasic and two grams of alpha-tricalcium phosphate powder to the solution and stir continuously with a spatula to combine. Add a 25 millimeters zirconia ball, secure the lid and place it inside a planetary mill for 60 minutes at 180 rotations per minute, pausing halfway to scrape down the sides of the jar with a spatula using a spatula.
Load the ink into a one milliliter syringe. Make a 10%solution of gelatin type A in 1X PBS as described in the text manuscript. Place the conical flask on the stirrer.
Then add 5.796 milliliters of methacrylic anhydride and continue stirring in the dark at 50 degrees Celsius for 90 minutes. Quench the reaction by diluting the conical flasks contents two fold with PBS. Decant into 50 milliliter tubes and remove the unreacted methacrylic anhydride by centrifugation at 3000 RCF at room temperature for three minutes and collecting the supernatant.
Dialyze the supernatant inside 14 kilodalton cutoff cellulose dialysis tubes against deionized water at 40 degrees Celsius for five days while gently stirring. Replace the deionized water every day. Prepare for storage by decanting into 50 milliliter tubes, securing the cap and placing it in the refrigerator for 12 hours.
Then freeze the samples using liquid nitrogen and immediately lyophilize for five days at minus 54 degrees Celsius and 0.4 millibars. Store the resulting foam in a freezer at minus 20 degrees Celsius. To synthesize GelMA microgels, make a 10%weight by weight GelMA solution in PBS by weighing the lyophilized GelMA, adding it to a tube with PBS and heating in a water bath at 50 degrees Celsius until fully hydrated.
Add 37 milliliters of oil per one milliliter of GelMA solution to a beaker, ensuring it is no more than 65%full. Set up a double beaker system on a hot plate with magnetic stirring by placing the beaker containing oil inside a larger beaker. Heat to 40 degrees Celsius while stirring.
Load the GelMA solution into a syringe and add it dropwise into the stirring oil through a 0.45 micrometer filter. Allow the emulsion to equilibrate for 10 minutes. Reduce the temperature of the emulsion to 15 degrees Celsius to thermally.
Stabilize the spheres by adding crushed ice into the space between the two beakers. Add acetone to the spinning GelMA emulsion. Decant the contents of the beaker into 50 milliliter tubes, making sure to wash the walls of the beaker with acetone.
Let it rest for 20 minutes to allow the dehydrated micro gels to settle to the bottom. Discard the supernatant and wash the microgels twice with acetone. Consolidate into one tube, top up with acetone and sonicate for 10 seconds.
Again, wash it with acetone twice. Store in acetone at room temperature until required for printing. To prepare the GelMA microgel suspension for printing, formulate a 1%weight by weight solution of GelMA in DMEM as described in the text manuscript.
Evaporate the acetone from the dehydrated microgels and weigh the resulting powder into a tube. Add acetone and transfer to a sterile environment. To form the microgel suspension, evaporate the acetone from the powder inside the biosafety cabinet.
After evaporation, add DMEM, 1%GelMA solution in DMEM and 2.5%LAP initiator solution to achieve a final packing fraction of 30%Allow to fully hydrate for at least 12 hours at room temperature. Culture the adipose-derived mesenchymal stem cells in low glucose DMEM supplemented with 10%FBS and 1%penicillin-streptomycin at 37 degrees Celsius and 5%carbon dioxide until confluent. Remove the adipose-derived mesenchymal stem cells from the tissue culture flask by removing the medium and washing with sterile PBS.
Pellet the cells by centrifuging at 150 RCF at room temperature for five minutes. Count the cells and calculate at least 5 x 10 to the five cells for each milliliter of GelMA microgels. Allocate the required volume of the cell suspension to a separate tube and pellet as demonstrated earlier.
Carefully remove as much supernatant as possible using a pipette, leaving only the cell pellet. Add the required volume of microgel suspension to the pellet and gently pipette up and down to ensure even distribution. Load the cell-laden in microgel suspension into a reactor using a pipette.
Using a one milliliter syringe fitted with a 23 gauge needle, deposit the bone-ink, crosslink the cell and bone-ink laden GelMA microgel constructs with a UV crosslinker lamp at 405 nanometers for 90 seconds. Immediately transfer the microgel suspension to an appropriately sized well plate containing complete DMEM. Incubate at 37 degrees Celsius and 5%carbon dioxide.
Replace the culture medium after 24 hours, then every 48 to 72 hours as required. The bone-ink was analyzed by scanning electron microscopy to determine its micro structure, which showed the formation of hydroxyapatite crystal after ink setting, particularly visible in dry conditions. In this study, cells were mixed with GelMA microgels and kept in culture for up to five days, either in micro gel suspension only or in the COBICS system.
The cells showed good viability in the presence of the bone-ink, both on day one and day five, confirming that the leaching products of the ink were not detrimental to the cells. It is crucial to control the stirrig speed while synthesizing microgel as a slight changes stirring can cause size differences. It is also important to ensure homogenous distribution of the cells within microgel suspension.
The same protocol can be used to print inside gelatin microgel suspensions, which act as sacrificial supports, leaving behind standalone bone constructs. But using GelMA opens up the avenue for one pot multiphasic constructs, such as osteochondral or bone tendon interfaces.
